Composition and method for affecting obesity and related conditions

ABSTRACT

A composition including an effective amount of a compound including manganese [III]tetrakis(4-benzoic acid)porphyrin (MnTBAP), derivatives or analogs thereof to induce a reduction in body weight of a mammal or to improve insulin sensitivity of an obese mammal. A method including introducing an effective amount of a compound including manganese [III]tetrakis(4-benzoic acid)porphyrin (MnTBAP), derivatives or analogs thereof to a mammal to induce a reduction in body weight or to induce an increase in metabolism or insulin sensitivity of the mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 12/352,438, filed Jan. 12, 2009, which application claimspriority from U.S. Provisional Patent Application Ser. No. 61/041,885,filed on Apr. 2, 2008.

BACKGROUND

1. Field

Compositions and methods for affecting obesity and obesity relatedconditions.

2. Background

Obesity generally refers to a condition in which there is an excessaccumulation of body fat resulting in an increased body weight and bodyfat percentage. Obesity has been recognized as a chronic metabolicdisorder associated with numerous other maladies. For example, obesityis associated with increased instances of complications such ashypertension, type 2 diabetes, arteriosclerosis, dyslipidemia, certainforms of cancer, sleep apnea, and osteoarthritis.

The causes of obesity are not well understood on a biochemical level,but chronic positive energy balance, a state in which caloricconsumption exceeds caloric expenditure, leads to the accumulation ofbody fat. Methods for treating and managing obesity have therefore beendirected towards decreasing food consumption and increasing energyexpenditure. Identifying, characterizing, and developing chemical agentsfor these purposes gained much momentum after the discovery of leptin, a16 kilodalton (kDa) protein hormone that plays a key role in regulatingenergy intake and energy expenditure by decreasing appetite andincreasing metabolism. Since a relatively small percentage of humanobesity is due to leptin deficiency, alternative biochemical approacheshave been the subject of intense research. Unfortunately, many, if notall, of the drugs that have been identified tend to induce undesirableside effects such as dry mouth; anorexia; constipation; insomnia;dizziness; nausea; and, in the case of the medication fenfluramine ordexfenfluramine and phentermine (Fen-Phen), heart-valve complicationsthat can lead to death.

BRIEF DESCRIPTION OF THE DRAWING

The embodiments herein are illustrated by way of example and not by wayof limitation in the FIGURE of the accompanying drawing in which likereferences indicate similar elements. It should be noted that referencesto “an” or “one” embodiment in this disclosure are not necessarily tothe same embodiment, and such references mean at least one.

FIG. 1 illustrates a method for reducing mammalian body weight.

DETAILED DESCRIPTION

Obesity is a serious and growing public health problem that isassociated with many health related conditions, as previously discussed.Furthermore, insulin resistance has been found to be associated withboth obesity and obesity related conditions such as type 2 diabetes,hypertension and hyperglycemia, to name a few.

Insulin is a hormone secreted by the pancreas that notifies cells toimport glucose. Insulin causes liver and muscle cells to take in glucoseand store it in the form of glycogen and causes fat cells to take inglucose and store it in the form of triglycerides. Insulin resistancerefers to a diminished capacity in some tissues to respond to insulin.In this aspect, normal amounts of insulin are inadequate to produce anormal insulin response from fat, muscle, and liver, three of the mostmetabolically active organs in the human body. Insulin resistance isassociated with obesity and many of the above-described obesity relatedconditions. Accordingly, a treatment agent is disclosed herein withproperties that induce weight loss and increase insulin sensitivity fortreatment of obesity and obesity related conditions.

FIG. 1 illustrates a method for reducing mammalian body weight. Method100 includes identifying an obese mammal (block 102). Once the obesemammal is identified, a treatment agent such as a compound including aporphyrin is administered to the mammal to affect obesity and obesityrelated conditions (block 104). In some embodiments, the treatment agentaffects obesity by inducing weight loss in the mammal. In still furtherembodiments, the treatment agent affects obesity related conditions byincreasing insulin sensitivity in the mammal. The term obesity isgenerally used herein to refer to a condition in which there is anexcess accumulation of body fat resulting in an increased body weightand body fat percentage in a mammal (e.g. a human).

In one embodiment, the treatment agent may be a compound including aporphyrin. Representatively, the porphyrin may be a metal porphyrinincluding, but not limited to, MnTBAP. MnTBAP refers to manganese [III]tetrakis (4-benzoic acid) porphyrin. Another commonly used name forMnTBAP is manganese [III] tetrakis (5, 10, 15, 20 benzoic acid)porphyrin. In its solid state, MnTBAP is a chloride salt, but in aqueousand biological conditions, MnTBAP is a positively charged compound.MnTBAP, which does not occur naturally in mammalian cells, is a smallsynthetic molecule that does not cross the blood-brain barrier. It isbelieved that MnTBAP can catalyze the conversion of superoxide to a lessreactive molecule, hydrogen peroxide. In addition, MnTBAP scavengesperoxynitrite and strongly inhibits iron-mediated lipid peroxidation.Peroxynitrite is a highly reactive molecule associated with manydiseases such as type 2 diabetes. Although MnTBAP is identified,derivatives or analogs of MnTBAP are also contemplated, including saltforms that, for example, yield the positively charged manganeseporphyrin in biological conditions or compounds that are functionallysimilar to the positively charged porphyrin. In still furtherembodiments, analogs may include a manganese porphyrin related toMnTBAP, for example, Mn [III] tetra(4-pyridyl) porphyrin (MnTyP).Analogs may further include copper containing porphyrins, such ascopper(II) (3,5-diisopropylsalicylate)₂ (CuDIPS) and its derivatives.

The following experimental results illustrate MnTBAP's effectiveness atinducing weight loss and enhancing insulin sensitivity in a mammal.

EXPERIMENT I

The following experiment involved a diet-induced mouse model of obesity.In particular, male C57BL/6J mice (these wildtype mice are geneticallydistinct from ob/ob and db/db mice and are not leptin-deficient) wereplaced on a high fat diet (HFD) or control diet (CON) at 8 wks of age,and MnTBAP or vehicle administration was initiated 5 months later, whenall mice were at a steady state body weight. Mice receiving the CON dietserved as lean control animals to those receiving the HFD diet. Each ofthe CON and HFD mice groups were then further divided into two groups.One of the CON and HFD groups was treated with 10 mg MnTBAP/kg of bodyweight for 30 days, and the other groups received a vehicle for 30 days.The term “vehicle” refers to the solvent used to deliver MnTBAP. In thisexperiment, MnTBAP was dissolved in 2% bicarbonate (pH 8.0) to a finalconcentration of 2 mg/mL. Vehicle-treated mice received a volume ofvehicle that would have been sufficient to deliver 10 mg of MnTBAP perkg of body weight were the compound present. MnTBAP was purchased fromA.G. Scientific Inc., San Diego, Calif. Elemental analysis and gaschromatography performed by A.G. Scientific indicated that the purchasedMnTBAP was >98% pure. The above-identified doses of MnTBAP and vehiclewere administered to the mice as daily intraperitoneal injections.

At the start of the treatment, there were no significant differences inbody weight between MnTBAP and vehicle groups within each diet (CON:p=0.99; HFD: p=0.77). In addition, HFD mice were dramatically heavierthan CON mice at the start of treatment (p<0.001).

Before and after treatment, insulin sensitivity (IS) was assessed usingan insulin-assisted glucose tolerance test (IA-GTT) after a 16-hourfast. Fasting body weights were measured at the end of each fast justprior to initiation of the IA-GTT. Approximately 5-7 days after thepost-treatment IA-GTT, mice were stimulated with insulin ten minutesprior to delivery of an anesthetic cocktail to induce deep anesthesiafor harvesting of quadriceps, tibialis anterior, soleus, gastrocnemius,subcutaneous white adipose tissue (SWAT), epididymal white adiposetissue (EWAT), and liver. In addition, SWAT and EWAT weights weremeasured immediately after harvest. Tissues were snap frozen in liquidnitrogen and stored at −80° C. until analysis. One quadriceps musclefrom each mouse was homogenized for biochemical analyses.

Fasting body weights of the vehicle-treated mice were not significantlydifferent pre-treatment vs. post-treatment (p>0.05). Fasting body weightafter treatment, however, was lower than before treatment in both HFDand CON mice that received MnTBAP.

In both CON and HFD mice, MnTBAP induced dramatic reductions in fastingbody weight (CON: <0.01; HFD: p<0.001), SWAT weight (CON: p<0.05; HFD:p<0.01) and EWAT weight (CON: p<0.01; HFD: p<0.01) compared to vehicle.These changes occurred without affecting daily caloric intake (CON:p=0.62; HFD: p=0.79).

Experiment I showed that HFD mice that received MnTBAP treatment weighed18.6% less than HFD mice that received vehicle alone. CON mice thatreceived MnTBAP treatment weighed 12.4% less than CON mice that receivedvehicle alone. These comparisons were made after treatment and werestatistically significant (P<0.01). Moreover, in the fed state, i.e. notfasted, significant weight loss was observed after as little as 15 daysof treatment in HFD mice and as little as 27 days of treatment in CONmice. In HFD mice dramatic weight loss was seen after 30 days oftreatment.

It was further found that MnTBAP significantly enhanced insulinsensitivity in HFD mice (p<0.05).

Although 10 mg/kg of body weight of MnTBAP was administered in theforegoing experiment, it is contemplated that an effective amount ofMnTBAP may be anywhere from 2.5 mg/kg of body weight to 10 mg/kg of bodyweight. Doses up to 10 mg/kg of body weight have no known adverse sideeffects and pharmacotoxicity studies indicate that MnTBAP treatment upto a dose of 88 mg/kg of body weight in mice does not increase mortalityrates or decrease age of death. Thus, in still further embodiments, aneffective amount of MnTBAP may be from 2.5 mg/kg of body weight to lessthan 88 mg/kg of body weight.

Fasting body weights; fed-state body weights; and plasma glucose levelsat various times during the IA-GTT, a test used to assess insulinsensitivity, were compared after treatment using a 2×2 analysis ofvariance (ANOVA) with repeated measures. Here “repeated measures” refersto the time of glucose measurement during the IA-GTT: 0, 15, 30, 45, 60min. Subcutaneous white adipose tissue weights; epididymal white tissueadipose weights; caloric consumption; and all biochemical data, such asUCP-2, UCP-3, and PKB expression, were compared using a 2×2 ANOVAwithout repeated measures.

The weight loss observed in the above experiment was surprising andunexpected. In particular, various experiments have been performed inconnection with MnTBAP and its effects on mice. In the known studies,however, it was found that treatment of mice with MnTBAP induced eitherno change in weight or slowed weight gain but did not induce weightloss. In addition, in many of these studies, mutant mice that do notexpress leptin (“ob/ob mice”), a protein hormone that regulates satiety,were used. Since leptin is not present in ob/ob mice, these mice eatincessantly and become extremely obese and insulin resistant. Inducementof obesity in this manner is different from the embodiments disclosedherein in which wild type, i.e. normal or non-mutant, mice are used andobesity is induced using a high fat diet. In addition, the mice used inthe previous experiments were younger and received MnTBAP treatment muchearlier in life than those of Experiment I. In some cases, the previousstudies treated mice at only 8 weeks of age.

In analyzing the cause of such significant weight loss, it wasrecognized that approximately 50% of the weight loss was due todecreased lipid and subcutaneous fat deposits in just subcutaneous whiteadipose tissue (SWAT) and epididymal white adipose tissue (EWAT).Although these fat pads are prominent in mice, they represent only aportion of the total fat in a mouse; therefore, it is recognized thatthe weight loss was disproportionately localized in fat. In this aspect,and in light of the fact that food consumption did not change afterMnTBAP administration, it is contemplated that MnTBAP caused an increasein metabolism that subsequently led to weight loss.

In support of this contemplation, administration of MnTBAP (10 mg/kg)daily for four weeks resulted in increases in the expression ofuncoupling protein-3 (UCP3) in skeletal muscle of HFD mice (quadriceps,p<0.05). UCP3 expression was ˜60% greater in HFD mice treated withMnTBAP than those treated with vehicle. The UCP3 levels are highlycorrelated to changes in body weight (R=0.7, P=0.0004) and thereforeindicate that changes in uncoupling protein expression, particularlyUCP3 expression, play a role in MnTBAP-induced weight loss. UCP3 acts asan uncoupler of mitochondrial respiration. In other words, it disruptsthe electrochemical gradient established in the mitochondrialintermembrane space and thereby decreases the efficiency with which ATPcan be generated. As a result, more energy is required to generate ATPand whole body metabolism increases. In this aspect, it is believed thatthe weight loss is, at least in part, caused by the resulting increasesin UCP3 expression.

In particular, it is believed that the effect of MnTBAP treatment onskeletal muscle UCP3 expression may be indirect and mediated by anincrease in free fatty acids. Since mice treated with MnTBAP experienceda large decrease in both subcutaneous and epididymal white adiposetissue weights, it is believed that the free fatty acids liberated fromthese abundant lipid stores played a role in the increases in UCP3expression, particularly since it is known that infusions of free fattyacids increase UCP3 mRNA levels. Moreover, it is believed that theincrease in skeletal muscle UCP3 expression facilitates catabolism ofliberated free fatty acids. The lack of a significant increase in UCP3levels in CON mice treated with MnTBAP may be associated with theirsubstantially lower fat deposits from which to release free fatty acids.The observation further supports the conclusion that free fatty acidshave a role in the induction of UCP3 expression in skeletal muscle. Itis noted that if the presence of increased free fatty acids in and ofthemselves mediate the increase in UCP3 expression, then it would beexpected that a HFD would increase UCP3 expression. A significantincrease in UCP3 levels in muscles of mice fed a HFD compared to a CONdiet, however, was not found in Experiment I, a result that indicatesthat the dietary lipids are directed to storage rather than signalingtoward UCP3 induction. The results of Experiment I therefore support theconclusion that UCP3 expression mediates significant reductions in bodyweight, subcutaneous white adipose tissue, and epididymal white adiposetissue. In addition, the fact that caloric intake did not change in themice treated with MnTBAP provides further evidence that the increase inUCP3 plays an important role in the reductions of body weight and fatdepots.

In addition to affecting weight loss, MnTBAP has been shown by the aboveexperiment to increase insulin sensitivity. Increased insulinsensitivity can have beneficial effects on a variety of obesity relatedconditions. In particular, administration of MnTBAP (10 mg/kg) daily forfour weeks resulted in significantly improved insulin sensitivity and a˜60% increase in the activity and expression of protein kinase B (PKB)in skeletal muscle (quadriceps) of HFD mice treated with MnTBAP ascompared to HFD mice treated with vehicle. PKB is an important insulinsignaling molecule in skeletal muscle and other metabolically activetissues, such as adipose (fat) and liver. It was found that theincreased activity (i.e., phosphorylation) was due mainly to increasedexpression of the protein (i.e., there is more of the protein to bephosphorylated) (P<0.05). A statistically significant increase in PKBexpression was not observed in CON mice treated with MnTBAP. Theincreased PKB activity in HFD mice is sufficient to improve insulinaction in skeletal muscle, which is one of the primary sites for glucosemetabolism in mice and humans. This data supports the physiologicalresults, which show a statistically significant treatment effect onwhole body insulin sensitivity in HFD mice, as assessed by insulinassisted glucose tolerance tests (p<0.01). It is noted that HFD micewere very insulin resistant before treatment (p<0.001), but a maineffect for diet after treatment (p=0.3) was not detected, a result thatunderscores MnTBAP's effect on insulin action. This indicates thatMnTBAP treatment completely reversed or ameliorated the effect of a highfat diet on insulin sensitivity.

It is therefore contemplated that in addition to affecting obesity inmammals, administration of MnTBAP may be used to treat or prevent theonset of obesity-related and insulin resistance-relateddiseases/conditions by ameliorating obesity and insulin resistance.Examples of such diseases/conditions include type 2 diabetes (andtherefore its comorbidities, e.g. renal failure), hypertension,hyperglycemia, arteriosclerosis, and other forms of heart disease.

In one embodiment, the treatment agent is administered to a mammal inthe form of a composition. The composition may include the treatmentagent and a vehicle. The composition may be administered to the mammalby any technique suitable for introducing the treatment agent to themammal. Representatively, the composition may be administered via anoral, intravenous, rectal, transmucosal, intestinal, parenteral,intrathecal, intraventricular, intraperitoneal, transdermal,subcutaneous capsular dispersion (implant), intranasal or intraocularadministration route. The vehicle may be, for example, saline or anyother similarly suitable fluid for facilitating administration of thetreatment agent to the mammal via injection or some other means.Alternatively, the composition may be suitable for oral administrationin a solid or liquid form and may further include excipients such assugars or cellulose preparations and colorants.

The composition may be formed by mixing an amount of the treatment agentwith a vehicle until a concentration of the treatment agent effectivefor inducing weight loss and/or increasing insulin sensitivity in amammal is reached. Representatively, the composition may include 10 mgof MnTBAP per kg of body weight of the mammal. In still furtherembodiments, the composition may include from about 2.5 mg/kg of bodyweight to about 10 mg/kg of body weight of MnTBAP.

The composition may be administered by any of the above routes pursuantto a regimen for administering the compound which induces a reduction inbody weight. Representatively, the composition may be administered tothe mammal periodically. In some embodiments the composition isadministered to the mammal once a day. In other embodiments, thecomposition is administered to the mammal once a day or more. Forexample, the composition may be administered twice daily or once a weekor once a month. It is contemplated that the frequency and duration ofadministration of the composition may vary depending upon the amount oftreatment agent in the composition and the desired effects.

In some embodiments, the composition is in the form of a pill, acapsule, a tablet, or a lozenge. The composition may further beadministered in the form of a powder. In other embodiments, thecomposition is administered in the form of an aqueous solution. In someembodiments, the treatment agent in the form of a powder or aqueoussolution may be incorporated into a candy bar, food bar, or power baralong with substances typically used in those items, such as grains,fruits, flavorings, nuts, binders, etc. In still further embodiments,the composition is administered in the form of an implant implantedwithin the mammal which releases a desired amount of the treatment agentover time. It is contemplated that the form of the composition may varydepending upon the desired administration route. Representatively, wherethe composition is to be injected into the tissue of the mammal, thecomposition may be in the form of an aqueous solution.

In some embodiments, a release rate of the treatment agent into themammalian system may be controlled. In some embodiments, an entericcoating, a pH dependent polymer, a polymer in a matrix, or a delayedrelease coating may be used to control a release rate of the treatmentagent within the system. In some embodiments, the delayed releasecoating may be Eudragit S, a pH dependent copolymer of methacrylic acidand methyl methacrylate acrylic polymer that can be used to make delayedrelease pills, either as a component of the matrix in which thetreatment agent is mixed or as a coating on the outside of the pill orcapsule. The coatings or polymers may allow for a slow or sustainedrelease of the treatment agent. In some embodiments, the coatings orpolymers allow for immediate-release or periodic-release of thetreatment agent.

Although Experiment I describes results in connection with a mouse modelof obesity, it is widely recognized and widely used as the mostphysiologically representative experimental model for human obesity. Inthis aspect, it is contemplated that the affects on obesity achievedusing MnTBAP in the mouse model may further be achieved in humans.

It should be appreciated that reference throughout this specification to“one embodiment” or “an embodiment” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention.Therefore, it is emphasized and should be appreciated that two or morereferences to “an embodiment” or “one embodiment” or “an alternativeembodiment” in various portions of this specification are notnecessarily all referring to the same embodiment. Furthermore, theparticular features, structures or characteristics may be combined assuitable in one or more embodiments of the invention.

In the foregoing specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes can be made thereto withoutdeparting from the broader spirit and scope of the invention as setforth in the appended claims. The specification and drawing are,accordingly, to be regarded in an illustrative rather than a restrictivesense.

What is claimed is:
 1. A method of inducing an increase in metabolicrate in a mammal in need thereof comprising administering an effectiveamount of manganese [III] tetrakis (4-benzoic acid) porphyrin (MnTBAP)or analogs thereof.
 2. The method of claim 1 wherein an effective amountof compound is an amount which further enhances insulin sensitivity. 3.The method of claim 1 wherein an effective amount of the compound isfrom about 2.5 mg/kg of body weight about 88 of body weight of themammal.
 4. The method of claim 1 wherein an effective amount of thecompound is Introduced to the mammal on a daily basis.
 5. The method ofclaim 1 wherein an effective amount of the compound is an amount whichfurther treats type 2 diabetes.